Systems and methods for bio-inactivation

ABSTRACT

A system for irradiating a microplate may include a modular light engine with one or more light emitting devices. The light emitting devices are configured to emit germicidal radiation to irradiate the microplate, which is configured to be positioned below the modular light engine inside a chamber of the microplate irradiation system. In this way, a uniform intensity of germicidal radiation may be output by light emitting devices, resulting in disruption of contaminating nucleic acids present in the microplate.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 62/412,030, entitled “MICROPLATE IRRADIATION SYSTEM”, filed on Oct.24, 2016, the entire contents of which are hereby incorporated byreference for all purposes.

BACKGROUND/SUMMARY

Clinical and laboratory settings require sterilization of equipment toeradicate microorganisms, protein, and/or nucleic acid contaminants.Methods for inactivation of biological microorganisms and contaminantscurrently in use are either chemical-based or illumination-based. Whilemany microorganisms and contaminants are easily removed or inactivatedusing standard chemical disinfection methods (e.g., bleach, alcohol);some microorganisms are resistant to standard sterilization techniques.Additionally, some enzymes found in living cells and biological fluidsmay act as molecular contaminants (e.g., ribonuclease enzymes or RNaseA), and may be highly resistant to denaturation. Furthermore,chemical-based disinfection methods may not be suitable for use withcertain surfaces/materials and may not affect irreversible inactivation.

The inventors herein recognize the forgoing issues and propose methodsto partially address them. In an example approach, a microplateirradiation system includes a bio-inactivation device comprising amodular light engine with one or more light emitting devices, where eachlight emitting device may include an array of light emitting diodesconfigured to emit germicidal radiation. In one embodiment, thegermicidal radiation may be directed towards a microplate inserteddirectly below the modular light engine inside a chamber of thebio-inactivation device. The microplate may contain a liquid reagentmixture and when positioned below the modular light engine, may receivea uniform intensity of germicidal radiation (e.g., ultraviolet light)from the array of light emitting diodes, resulting in disruption ofcontaminants present in the reagent mix.

In this way, reagents dispensed in a microplate may be sterilized byintroducing the microplate in a bio-inactivation device, including amodular light emitting engine with a plurality of light emitting diodesemitting germicidal radiation. Emission from the a modular light engineis incident on the microplate at a (relatively) uniform spatialintensity, which disrupts the contaminating nucleic acids in the reagentmix, making the reagent suitable for subsequent nucleic acid sequencingand/or amplification protocols.

In another example, a bio-inactivation device comprising a modular lightengine with one or more light emitting devices configured to emitgermicidal radiation may be configured as a composite hand-heldillumination unit for disinfection of surfaces (e.g., lab benches andhoods), glassware, etc., without the microplate attachment. Herein, thebio-inactivation device may be configured to emit germicidal radiationdirected towards surfaces and materials to be treated (e.g.,disinfected). The spatio-temporal emission pattern from the hand-heldbio-inactivation unit may be regulated by a controller either manually(by user input) or automatically based on feedback from one or moresensors in the bio-inactivation unit. In one example, the unit mayinclude a closed loop control system using a photodetector to measurereflectance from the surface before and after treatment. In anotherexample, the closed loop control system comprising the photodetector maymeasure fluorescence of the surface before and after treatment. In theembodiments described above, an inactivation unit may be used to emitgermicidal radiation of a single specific wavelength that effectivelytargets biological organisms and/or targets contaminating nucleic acidsby disrupting their DNA structure. Alternatively, the device may outputa combination of two or more wavelengths to enable permanent andcomplete inactivation by targeting different aspects of the organisms.

In this way, a compact bio-inactivation unit combined with aphotodetector may be used as a point-of-use device to treat surfaces fordisinfection. The device may initially measure the surface reflectanceand/or irradiance prior to germicidal radiation, followed by exposure togermicidal radiation of one or multiple wavelengths and subsequentre-measurement of the surface reflectance and/or fluorescence todetermine the level of inactivation efficacy. A more complete andefficient inactivation of biological contaminants may therefore beachieved using multiple wavelengths of light with concurrent real timefeedback based on the change in surface reflectance and/or fluorescenceproperties.

The advantages and features of the present description are captured inthe following detailed description; either singularly or in connectionwith the accompanying drawings.

It is to be understood that the summary above is provided to introduce asimplified selection of concepts that are described further in thedetailed description. It is not meant to identify key or essentialfeatures of the claimed subject matter, the scope of which is defineduniquely by the claims that follow the detailed description.Furthermore, the claimed subject matter is not limited toimplementations that solve any disadvantages noted above or in any partof this disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a schematic of a first embodiment of abio-inactivation unit in the form of a microplate irradiation system.

FIGS. 2-3 show the microplate irradiation system.

FIG. 4 shows a display screen of the microplate irradiation system.

FIGS. 5-6 show transparent views of the microplate irradiation system.

FIG. 7 shows air circulation through the microplate irradiation system.

FIGS. 8-9 illustrate a modular light engine of the microplatesterilizing system.

FIG. 10 illustrates a single light-emitting device of the modular lightengine.

FIG. 11 illustrates a light-emitting device inside a housing of themicroplate irradiation system.

FIG. 12 illustrates an operating method for the bio-inactivation devicewith the microplate irradiation system.

FIG. 13 illustrates a schematic of a second embodiment of abio-inactivation device.

FIG. 14 illustrates a schematic of a third embodiment of abio-inactivation device.

FIG. 15 illustrates a fluorescence-based method for determiningbio-inactivation level using multi-wavelength germicidal light

FIG. 16 illustrates a reflectance-based method for determiningbio-inactivation level using multi-wavelength germicidal light.

FIG. 17 shows a graph depicting RNase A enzyme activity at variedirradiance levels.

FIG. 18 shows a graph depicting RNase A enzyme activity with increasingexposure to 275 nm UV light at 50% relative irradiance level.

FIG. 19 shows a graph depicting the synergistic effect of using multipleUV light wavelengths on RNase A enzyme activity.

DETAILED DESCRIPTION

The present description relates to methods and systems for inactivationof biological organisms and other molecular contaminants, comprising abio-inactivation device with a modular light emitting engine andoptionally with a cavity/drawer configured to house a microplate, forsterilizing reagents and surfaces by radiation, such as UV-C radiation.

As described above, sterilization of clinical and/or laboratory settingsmay include chemical- or illumination-based techniques that caninsufficiently target desired organisms or enzymes. These techniques maysuffer from additional issues. For example, illumination-based methodsof inactivation may employ conventional lamps (e.g., gas-discharge lampsor mercury arc), which may afford higher efficacy than chemical methods,but may be incapable of determining the efficacy of bio-inactivation.Additionally, lamp-based UV systems may be expensive and cumbersome,have short lifetimes, and include a warm-up period to attain stableoutput, making them impractical for daily use.

An example approach involves UV germicidal irradiation as anillumination-based disinfection method that uses short-wavelength UV(i.e. UVC) light to inactivate microorganisms. Ultraviolet (UV)radiation ranges from 100-400 nm with four distinct spectral regionsincluding; vacuum UV (100-200 nm), UVC (200-280 nm), UVB (280-315 nm),and UVA (315-400 nm). A mechanism of UVC inactivation of biologicalmicroorganisms is cellular damage caused by disruption (distortion) oftheir nucleic acid structure when UVC is absorbed. The UVC spectrum,especially in the range of 250-270 nm with 265 nm being the peakgermicidal wavelength, is commonly known as germicidal UV as it isstrongly absorbed by the nucleic acids of an organism.

Ultraviolet irradiation is used for sterilizing laboratory equipment andreagents, disinfecting surfaces and materials, wastewater treatment, airdisinfection, and for disinfection of various home devices fromtoothbrushes to tablet computers. There is an increasing demand inlaboratories for methods that ensure the purity of DNA samples used astemplates and reagents employed, especially for large-scale, automatedgenome analyses (e.g., high throughput amplification and sequencingmethods). Reagents may include nucleic acids from contaminatingmicroorganisms, human DNA introduced as a contaminant (while handlingthe reagents), microorganisms as contaminants which release their DNAwhen inactivated, and/or enzymes that exhibit activity even in theabsence of nucleic acids or live microorganisms (e.g., an RNA librarypreparation for sequencing is an example). For example, touchcontamination from human skin may include enzymes as well asmicroorganisms and shed human cells. Such contaminants, even if presentin trace amounts, may interfere with nucleic acid amplification andaffect the fidelity of sequencing. Thus, during the preparation ofreagents for sequencing (e.g., for DNA sequencing using microfluidictechnologies), sterilization of the reagents to free the reagents ofunwanted contaminants (i.e. nucleic acids, various proteins, andmicroorganisms) may help eliminate false positive results, increase thesignal-to-noise ratio, and generate reproducible and accuratehigh-throughput sequencing data. Cartridges used in preparation for DNAlibrary sequencing, which are often contaminated through differentsources, may be rendered contaminant-free by exposure to UV.

However, more resistant organisms may survive the single wavelengthgermicidal UVC exposure and multiply. Some organisms may even reactivateover time following UV inactivation, rendering the exposure ineffective.Further, the UV illumination of such large DNA sequencing chambers usinggas-discharge lamp systems may not be uniform, which can increasesterilization times, energy consumption, and operating costs. Moreover,any contaminating microorganisms present in the reagent mix or presenton surfaces in contact with the reagent mix may be lysed by the hightemperatures used during the sequencing protocols, and may contributeadditionally to the contaminating nucleic acids. The partiallyinactivated contaminating nucleic acids in the reagents or presence ofmicroorganisms on surfaces and materials (e.g., tubes, plasticware), mayinterfere detrimentally with laboratory protocols, including DNA and/orRNA sequencing and amplification.

Thus, according to embodiments disclosed herein, the exposure ofbiological contaminants to select multiple wavelengths of light mayyield complete and effective bio-inactivation of reagents and surfacesalike. Further, a multi-wavelength targeted approach may irreversiblyinactivate microorganisms and other contaminants such as enzymes,preventing the issue of reactivation of microorganisms over time whentreated with UV light of a single wavelength.

FIG. 1 illustrates a schematic of a first embodiment of abio-inactivation unit in the form of a microplate irradiation system. Amicroplate may be inserted within a chamber of the bio-inactivationunit, which includes the modular light-emitting engine, as shown inFIGS. 2-3. The microplate may be irradiated by activating the modularlight-emitting engine through a menu on a display screen as shown inFIG. 4, where the display screen is coupled to a controller. FIGS. 5-6illustrate the configuration of various components inside the microplatehousing. The modular light emitting engine may be coupled to acontroller and to a power supply and may be cooled by a ventingmechanism as illustrated in FIG. 7. The modular light emitting enginemay include a plurality of light emitting devices, wherein each devicemay include an array of light emitting diodes emitting germicidalradiation (e.g., UV-C), as illustrated in FIGS. 8-11. A duration,intensity, and pattern of irradiation may be regulated by thecontroller, to sterilize reagents contained in the microplate insertedinto the bio-inactivation device via the method illustrated in FIG. 12.

In a second embodiment, a bio-inactivation device may be configured as acompact hand-held unit and may be used for the disinfection of surfacesand materials. The hand-held bio-inactivation device may be coupled to aphotodetector which may determine a level of inactivation achieved basedon emitted fluorescence as depicted in FIG. 13. Fluorescence levels maybe measured prior to and after treatment of the surface with germicidallight. The emitted fluorescence from the treated surface may be comparedto a threshold value and a level of inactivation may then be assessed asillustrated by the method of FIG. 15.

In a third embodiment, a bio-inactivation device may be a hand-held unitfor surface disinfection and may further be coupled to a photodetector.Herein, the photodetector may determine a level of inactivation achievedbased on a change in reflectance from the untreated vs. the germicidallight treated surface as shown by the depiction of FIG. 14. The changein reflectance may be evaluated against a threshold and a degree ofinactivation may be assessed as illustrated by the method of FIG. 16.

In order to assess the viability of the bio-inactivation device foreffective disinfection of surfaces, a commonly found and easilymeasurable contaminant of the surface may be tested for inactivation. Anexample of a ubiquitous molecular contaminant present in living cellsand frequently found on surfaces in the laboratory includes theribonuclease enzyme protein RNase A. RNase A is highly resistant todenaturation (disruption of protein) and thus serves as an optimaltarget for the bio-inactivation device. FIG. 17 shows RNase A enzymeactivity measured as relative fluorescence, when the surface containingRNase A is treated with UV light of 275 nm wavelength at variousintensities (irradiance) for a specific duration. FIG. 18 shows RNase Aenzyme activity when the surface with RNase A is treated with UV lightof 275 nm wavelength at 50% irradiance over increasing durations ofexposure to UV.

Most inactivation methods and systems employ a single wavelength of UVlight optimal for inactivation of specific targets. However, byemploying two or more wavelengths of light targeting differentstructures or pathways within organisms, a synergistic effect may beobserved leading to a complete and efficient inactivation as depicted byFIG. 19. Regarding FIGS. 2-11, parts and features introduced once indescription with reference to a figure may not be reintroduced and/orre-described with reference to subsequent figures and may be referred toby the same number.

Referring now to FIG. 1, a block diagram for an example configuration ofa bio-inactivation device in the form of a microplate irradiation system10 is illustrated. The microplate irradiation system 10 may be used toemit light, such as visible light, UV light, infrared light, and/orother types of radiation. In one example, microplate irradiation system10 may comprise a modular light engine 12, a controller 14, and a powersource 16.

The modular light engine 12 may include a plurality of semiconductordevices 19, as illustrated in FIG. 1, however in other examples, themodular light engine may include a single semiconductor device. Each ofthe plurality of semiconductor devices 19 may include an array 20 oflight emitting diodes (LEDs), for example. In other examples, eachsemiconductor device may be an organic LED (OLED), laser diode, plasmadischarge, or other light source. In one example, the array 20 may be atwo-dimensional array of light emitting diodes. Semiconductor devices 19may provide radiant output 24. In one example, the radiant output 24 maybe UV-C radiation. The radiant output 24 may be directed to a microplate26 positioned inside a drawer 25 inserted into a housing 11 of themicroplate irradiation system 10. Returned radiation 28 may be directedback to the modular light engine 12 from the microplate 26 (e.g., viareflection of the radiant output 24). Some of the radiation output 24may be returned back from the microplate 26 while some of the returnedradiation may be from structures not directly in-line with the pluralityof semiconductor devices emitting the radiation. An intensity of theradiation may be relayed to the controller 14. Based on the intensity ofthe returned radiation relayed, the controller may regulate theintensity of the radiant output 24 of the plurality of semiconductordevices 19.

The radiant output 24 may be directed to the microplate 26 via couplingoptics 30. The coupling optics 30, if used, may be variouslyimplemented. As an example, the coupling optics may include one or morelayers, materials or other structures, such as flat windows, balllenses, light guides, etc., interposed between the semiconductor devices19 and the microplate 26, and providing radiant output 24 to surfaces ofthe microplate 26. The coupling optics 30 may be made from UVtransparent materials such as fused silica, fused quartz, or otherglass, silicone, polymers, or other materials.

Each of the layers, materials or other structure of coupling optics 30may have a selected index of refraction. By selecting each index ofrefraction, reflection at interfaces between layers, materials, andother structures in the path of the radiant output 24 (and/or returnedradiation 28) may be selectively controlled.

The plurality of semiconductor devices 19 may be coupled to thecontroller 14 via coupling electronics 22. The controller 14 may also beimplemented to control the semiconductor devices, e.g., via the couplingelectronics 22. The controller 14 may be connected to the power source16 and may be implemented to modulate power supplied from the powersource 16. Moreover, the controller 14 may receive data from the powersource 16. In one example, the irradiance at one or more locations atthe microplate 26 surface may be detected by sensors (for example,sensors along the surface of the microplate 26, adjacent to the surfaceof the microplate 26, and/or via returned irradiance 28) and transmittedto controller 14 in a feedback control scheme.

In addition to the power source 16 and the modular light engine 12, thecontroller 14 may also be connected to user interface 23. The userinterface 23 may include a keyboard, mouse, display, and/or a touchscreen display with a programmable menu, the programmable menu includingduration of irradiation, intensity and dose of irradiation, and patternof irradiation (that is, which of the semiconductor devices of theplurality of semiconductor devices will be operated at a given time).The controller 14 may also communicate to an external device 34 throughone or more ports of the microplate irradiation system, such as USBport, LAN port, etc. The data received by the controller 14 from theuser interface and/or the external device may be stored in a memory ofthe controller 14 and may be used to perform a programmed irradiationcycle.

The controller 14 may receive data of various types from one or more ofthe power source 16, the modular light engine 12, the external device34, and/or the user interface 23. As an example, the data may berepresentative of one or more characteristics associated with coupledsemiconductor devices 19. As another example, the data may berepresentative of one or more characteristics associated with therespective modular light engine 12, power source 16, user interface 23,and/or external device providing the data. As still another example, thedata may be representative of one or more characteristics associatedwith the microplate 26 (e.g., representative of the radiant outputenergy or spectral component(s) directed to the microplate). Moreover,the data may be representative of some combination of thesecharacteristics.

The controller 14, in receipt of any such data, may be implemented torespond to that data. For example, responsive to such data from any suchcomponent, the controller 14 may be implemented to control one or moreof the power source 16, the modular light engine 12 (including one ormore such coupled semiconductor devices), etc.

Individual semiconductor devices 19 (e.g., LED devices) of the modularlight engine 12 may be controlled independently by controller 14. Forexample, controller 14 may control a first group of one or moreindividual LED devices to emit light of a first intensity, wavelength,and the like, while controlling a second group of one or more individualLED devices to emit light of a different intensity, wavelength, and thelike. The first group of one or more individual LED devices may bewithin the same array 20 of semiconductor devices, or may be from morethan one array of semiconductor devices. Array 20 may also be controlledindependently by controller 14 from other arrays of the modular lightengine. For example, the semiconductor devices of a first array may becontrolled to emit light of a first intensity, wavelength, and the like,while those of a second array in the modular light engine may becontrolled to emit light of a second intensity, wavelength, and thelike. Further, in some examples, a first subset of semiconductor devicesof array 20 may be controlled to emit light of a first intensity andfirst wavelength, while a second subset of semiconductor devices ofarray 20 may be controlled to emit light of a second intensity and/orsecond wavelength.

As described above, the microplate irradiation system 10 may beconfigured to receive the microplate 26 placed in the drawer 25 that maybe inserted inside housing 11 below the modular light engine. Themicroplate irradiation system 10 may also include a safety interlocksystem to activate and deactivate the modular light engine 12 if thechamber is closed and opened, respectively.

FIGS. 2 and 3 show a microplate irradiation system 100 (similar to themicroplate irradiation system 10 of FIG. 1), including a housing 103.The housing 103 includes a front face 114, perpendicular to a topsurface 102 and a bottom surface 119 of the housing 103. A back face 115of the housing is opposite to the front face 114, and is perpendicularto the top surface 102 and the bottom surface 119. The front face 114includes a drawer 104 that slides into and out of an opening 107 of thefront face 114. The housing 103 also includes a first side surface 112and a second side surface 113, opposite and parallel to the first sidesurface 112. Each of the first side surface 112 and second side surface113 may be perpendicular to the top surface 102 and bottom surface 119along a length L of the first side surface and the second side surface.The microplate irradiation system 100 may be a benchtop irradiationsystem and may be stackable.

The drawer 104 may be fully extended out of the opening 107 or may beinserted completely inside the opening 107, as shown in FIGS. 2 and 3,respectively. A length L1 of the drawer 104 may be less than the lengthL of the side surface, such that the drawer may fully insert into theopening 107. A mechanism 109 may be present on a drawer front 118 of thedrawer 104 to pull the drawer out of the opening 107 or to push back thedrawer into the opening 107. In one example, the drawer 104 may beinserted and extended out of the opening 107 using a switch (not shown),wherein the switch may be electrically operated.

A width W1 of a drawer front 118 may be more than a width W2 of theopening 107, such that a drawer front 118 of the drawer 104 is in facesharing contact with the front face 114 of the housing 103 around theopening 107 when the drawer is in a fully inserted position, asillustrated in FIG. 3.

The drawer 104 may include channels 105, which may slide intocomplementary grooves (not shown) inside the opening 107, thus enablingthe drawer to slide in and out of the opening 107. In one example, whenthe drawer is in the fully extended position out of the opening, thedrawer may not be detached from the housing 103. In another example, thedrawer 104 may be reversibly detached from the housing 103. In a furtherexample, a door (for example, a hinged door, a sliding door etc.)instead of the drawer may be configured to block and unblock the opening107.

The drawer 104 includes a cavity 117 with a stage 108. The stage 108 maybe configured to hold a reagent-holding device 106. In one example, thereagent-holding device 106 may be a multi-well microplate, for example,a 96 well plate or a 48 well plate. In another example, thereagent-holding device 106 may be one or more microfluidics chip devicesand/or cartridges. Other examples of the reagent-holding device 106 maybe a cuvette, a tissue culture flask, a slide, a tissue culture singlewell plate, etc. In a further example, the stage may be configured tohold more than one reagent holding device. The reagent-holding device106 may be reversibly fixed to the stage 108 (e.g., using one or moreclasps), such that sliding the drawer 104 in and out of the opening 107may not dislodge the reagent-holding device 106 from the stage 108.Additionally, the stage 108 with the reagent-holding device 106 may notinterfere with the drawer sliding in and out of the opening 107. In someexamples, the reagent-holding device 106 may include a lid comprised ofmaterial that is UV-transparent, such as clear silicone,polytetrafluoroethylene (PTFE), or fluorinated ethylene propylene (FEP).In such examples, the reagent-holding device may be inserted into thecavity/drawer with the lid coupled to the base of the reagent-holdingdevice. However, in examples where a lid of the reagent-holding deviceis not UV-transparent (e.g., lids comprised of polyester or polyimide),the lid may be removed prior to insertion into the cavity/drawer.

The front face 114 of the microplate irradiation system 100 alsoincludes a display screen 110 adjacent to the drawer 104, as illustratedin FIGS. 2-3. A magnified version of the display screen 110 is alsoillustrated in FIG. 4. In one example, the display screen 110 may be atouch screen. The display screen 110 may include a menu to operate themicroplate irradiation system 100. The display menu may be coupled to acontroller (for example, the controller 14 of FIG. 1), which mayregulate operation of a light source (such as time of light sourceactivation, intensity of the light source, etc.) as will be discussedfurther below. In other examples, the display screen may be positionedalong the top surface, or may be positioned along a first side surfaceor a second surface of the housing 103.

As shown in FIG. 4, the display screen may include user interfacecontrol elements that allow a user to input various parameters and/orcommands. For example, a user may select sterilization protocol (alsoreferred to as a recipe), which defines the light output parameters(e.g., power or irradiance level, emission pattern, temporal duration,wavelength spectrum). The user may also initiate activation andtermination of the sterilization procedure. The display screen may alsodisplay information regarding the sterilization procedure to the user,including exposure time, intensity level, selected recipe, microplateidentification, light emitter status, and drawer status.

A port 120 may be present along the front face 114 of the microplateirradiation system 100. In one example, the port 120 and/or additionalports may be present along other surfaces of the housing, such as alongthe first side surface and/or the second side surface or at the backface. The port 120 may be a wired and/or wireless communication means,such as a USB connection, a wireless internet connection, an infraredtransponder, or a Bluetooth® link. The microplate irradiation system 100may be connected through the port 120 directly and/or indirectly (viathe internet, an intranet, cellular network, PSTN or any other network)to an external CPU, such as a computer, having appropriate software toselect or configure one or more irradiation parameters and transfer themto a memory of a controller of the microplate irradiation system 100.Alternatively, the external device could be a memory store such as aROM, e.g., a USB stick, on which one or more light irradiationparameters are stored, which are transferred to the microplateirradiation system 100 controller memory upon connection.

FIGS. 5 and 6 show a first transparent view 200 and a second transparentview 201 respectively of the microplate irradiation system 100 describedabove with reference to FIGS. 2-3. The first transparent view 200 andthe second transparent view 201 illustrate the configuration ofcomponents inside the housing 103 of the microplate irradiation system100. Parts and features introduced previously in FIGS. 2-3 are notreintroduced and are referred to by the same number.

FIGS. 5 and 6 show transparent views of housing 103 with base supports230 along the bottom surface 119 of the microplate irradiation system.The reagent-holding device 106, for example, a microplate with a liquidreagent, may be in the drawer inserted inside the opening 107. A modularlight engine 220 may be positioned directly above the reagent-holdingdevice 106, such that light emitted from the modular light engine 220may be directed towards a top surface of the reagent-holding device 106.

The modular light engine 220 may be a non-limiting example of modularlight engine 12 of FIG. 1 and thus include a plurality of light sourcedevices (similar to the plurality of semiconductor devices 19 of FIG. 1)as will be described below with reference to FIGS. 7-11. The modularlight engine 220 may be coupled to a controller 218 (similar to thecontroller 14 of FIG. 1) inside the housing 103 of the microplateirradiation system 100. The controller 218 may be coupled to a powersource 240 (similar to the power source 16 of FIG. 1). In one example,the input power from the power source may be in the range of 90-260 VAC,47-63 Hz. The controller 218 may also be coupled to the display screen110 through coupling electronics 222 and may be coupled to the modularlight engine 220 through coupling electronics 224. The controller maycommunicate with the display screen 110 and with the port 120, asdescribed above with reference to FIG. 2.

Air vents 216 may be present along the back face 115 of the housing 103.The vents 216 enable the circulation of air through the housing 103 toreduce the temperature increase during operation of modular light engine220 inside the housing 103. In one example, a fan (not shown) may becontained within the housing (e.g., adjacent to the vents 216), tofurther aid in air-circulation through the housing 103.

A transparent side view 300 illustrating air circulation through thehousing 103 of the microplate irradiation system 100 is shown in FIG. 7.Air may enter the housing through the vent 216 along the back face ofthe housing 103. Cooler ambient air may flow through the vent 216 alongan airflow path 302 towards the front face 114 of the housing, flowingpast the controller 218, the coupling electronics 224, and the modularlight engine 220. The air temperature increases as heat is transferredconvectively while flowing past the controller, the electrical circuit,and the modular light engine. The heated air flows along a path 304 andexits the housing through the vent 216, thus reducing the internaltemperature of the housing while operating the modular light engine.

FIGS. 8-9 illustrate a modular light engine 400. Modular light engine400 is one non-limiting example of modular light engine 220 of FIGS. 2-3and 5-6 and modular light engine 12 of FIG. 1. The modular light engine400 may include a plurality of light emitting devices 420. In theillustrated example, each light emitting device 420 may include an arrayof LEDs arranged on a heatsink. In another example, the plurality oflight emitting devices 420 may be one or more tiles and/or strips, whereeach tile and/or strip may include a two-dimensional array of LEDsemitting radiation.

Each light emitting device 420 comprises an array of LEDs coupled to asubstrate. Each substrate is coupled to a heatsink, herein a set ofcooling fins. Each light emitting device 420 substrate forms the frontsurface of the light emitting device (e.g., light-emitting surface) andmay include a first end 422 and a second end 424 (opposite the first end422). The first end 422 and the second end 424 of each of the lightemitting device substrates may be in contact with the frame 404, while acenter section 426 (e.g., face) of each of the light emitting devicesfaces a reagent-holding device (e.g., device 106 of FIGS. 5-6). Light isemitted from each of the light emitting devices 420 unobstructed towardsthe reagent-holding device. The direction of light emitted from thelight emitting device is indicated by arrows 430. In one example, theframe 404 may be configured to accommodate up to seven light emittingdevices, as illustrated in FIG. 8. In other examples, the frame 404 maybe configured to accommodate more than seven or less than seven lightemitting devices. Fins 221 along a rear surface of each of the lightemitting devices 420 face away from the reagent-holding device and mayenable air circulation to reduce overheating of the modular lightengine.

In one example, the frame 404 may be directly above and correspond tothe stage 108 with the reagent-holding device (without being inface-sharing contact with the reagent-holding device) when the drawer104 is fully inserted in the opening 107, as illustrated in FIGS. 3 and5-6. The light emitted by the light emitting devices 420 may be directedto the top surface 208 of the reagent-holding device 106.

FIG. 9 shows a view 402 of a front surface 406 of the light emittingdevices 420. Each of the light emitting devices 420 includes a row ofLEDs 440 (while seven rows of LEDs are shown in FIG. 9, only one row islabelled in FIG. 9). The LEDs may emit UV, IR, and/or visible light. TheUV light wavelength may be in the range of 100 nm-290 nm (for example,275 nm), which is germicidal and can denature nucleic acids, includingthe contaminating nucleic acids present in the reagent inside thereagent-holding device. However, other wavelengths are possible,including multiple wavelengths, as described in more detail below.

FIG. 10 shows a top view 600 of the light emitting device 420, with thefirst end 422 and the second end 424. Each of the first end and thesecond end includes a retaining mechanism 446, which is configured tocouple each of the first end and the second end of the light emittingdevice 420 to the frame 404, as illustrated in FIGS. 8-9. The centersection 426 of the light emitting device includes a row of LEDs 436. Asshown in FIG. 10, the light emitting device 420 may include one row ofLEDs, where the row can accommodate up to four groups of LEDs, with upto eight LEDs in each group. However, the actual number of LEDs includedin the light emitting device may vary. In one example, the row of LEDs436 may include eight LEDs spaced apart from each other uniformly ornon-uniformly. In other examples, a different distribution of the LEDsin the center section 426 of the light emitting device may be seen, suchas more or fewer LEDs, more than one row, etc. The light emitting deviceshown in FIG. 10 may have a width of 23 mm, although other widths arepossible. Light emitting device 420 further includes a plurality offinger clips, such as clip 450, held down by screws, such as screw 452.The finger clips may establish electrical contact with the LEDs.

Each of the LEDs or groups of LEDs (i.e. rows, columns, or groups withinthe rows or columns), may be coupled to the controller and each LED orgroup of LEDs activated and deactivated by the controller. Thecontroller may regulate the power supply to the LEDs based on inputreceived from a user through the display screen 110 or through the port120, as described above. Each of the LEDs may emit light of the sameintensity, such that the reagent-holding device receives a uniform dose(for example, approximately 80% uniformity) of UV light on the topsurface of the reagent-holding device facing the LEDs. In one example,UV light emitted by the modular light emitting engine may have anapproximate intensity of 4.8 mW/cm² at the top surface of thereagent-holding device. The light generated is incident on thereagent-holding device, thus irradiating the reagent-holding deviceuniformly.

Each of the light emitting devices may be regulated individually by thecontroller. Similarly, the output of each of the LEDs or groups of LEDsmay be regulated by the controller. In one example, only a first sectionof the reagent-holding device may be irradiated by activating only thelight emitting devices corresponding to the first section. A secondsection of the reagent-holding device may receive no incident lightemission by not activating the light emitting devices corresponding tothe second section. In a further example, the light emitting devicescorresponding to the first section of the reagent-holding device may beoperated at a different intensity and/or a different temporal durationthan the light emitting devices corresponding to the second section ofthe reagent-holding device.

FIG. 11 shows a view 800, where one light emitting device 420 ispositioned inside the housing 103 of the microplate irradiation system100. The light emitting device 420 is positioned over a cavity 810. Thedrawer 104 is extended outside the housing 103. The stage 108 isconfigured to receive a microplate. When the microplate is positioned onthe stage and the drawer is inserted into the housing, the lightemitting device 420 is positioned over the microplate for deliveringgermicidal irradiation.

A method 1200 for operating a microplate irradiation system isillustrated in a flowchart in FIG. 12. In one example the method 1200may be used to operate the microplate irradiation system 10 and/ormicroplate irradiation system 100 illustrated in FIGS. 1-11.Instructions for carrying out the method 1200 may be executed by acontroller, for example, the controller 14 of FIG. 1 and/or thecontroller 218 of FIGS. 2-3, based on instructions stored in thecontroller memory and in conjunction with signals received by thecontroller from the display screen 110, the port 120, the modular lightengine 220 etc., illustrated in FIGS. 2-11.

The method 1200 begins by receiving an indication that a microplate hasbeen inserted into a chamber of the microplate irradiation system. Themicroplate may be positioned inside a drawer extending out of themicroplate irradiation system. The microplate wells may include liquidreagents. Hence, the microplate may be positioned on an even/flatsurface of the drawer, such as a surface of stage 108, with the wells ofthe microplate facing away from the even surface. After the microplateis positioned inside the drawer, the drawer may be inserted back into ahousing of the microplate irradiation system. In another example,reagent-holding devices such as microscopic slides, tissue cultureplates, etc. may be inserted into the housing by placing the devices inthe drawer. In another example, the microplate may be positioneddirectly inside the housing through an opening accessible through a doorcoupled to the opening. Once inside the housing, the microplate ispositioned directly below the modular light engine, such that themodular light engine may direct light emission to the microplate withoutany physical and optical obstruction between the microplate and themodular light engine. Once the microplate is positioned inside thehousing, ambient light and air flow through the opening is blocked, forexample, by inserting the drawer all the way into the housing. Thecontroller may receive an indication that the microplate has beeninserted inside the drawer and the drawer has been closed based on auser input and/or based on detecting that the drawer has opened and thenclosed.

At 1204, the method 1200 includes receiving parameters for anirradiation cycle for irradiating the inserted microplate. In oneexample at 1206, the parameters may be received via a user input, forexample the user may select the parameters using a display screen menucoupled to the microplate irradiation system and the selected parametersmay be relayed to a controller, such as the controller 14 of FIG. 1. Theselected parameters may include temporal duration, spatial pattern (byactivation of specific light emitting devices), intensity level, dose(power), etc. In another example at 1208, the irradiation parameters maybe received through a compatible external device, such as computer, aUSB drive, etc., that may be relayed through a port of the microplateirradiation system to the controller.

At 1210, method 1200 includes operating the microplate irradiationsystem as per as the selected parameters to irradiate the microplate andthe liquid reagents inside the microplate wells. The modular lightengine may be activated to deliver germicidal UV radiation to themicroplate at the selected intensity, dose, pattern, and duration. Asexplained previously, the modular light engine may include a pluralityof light emitting devices, such as LEDs. Thus, one or more of the LEDsmay be activated at a selected intensity, wavelength, and duration.Further, the selected pattern of light emission may be achieved byactivating different subsets of the LEDs at different times and/or withdifferent parameters. For example, all of the LEDs may be activated atthe same time, with the same or varying intensities, wavelengths, and/ordurations. In another example, a first subset of LEDs may be activatedat a selected intensity, wavelength, and/or duration, while a second,different subset of LEDs may be activated at a different intensity,wavelength, and/or duration.

After the irradiation cycle is complete, the method 1200 proceeds to1212 to eject the irradiated microplate from the housing of themicroplate irradiation system. The microplate may be ejectedautomatically upon completion of the irradiation cycle or upon receivinga user input requesting the microplate be ejected. To eject themicroplate, the drawer may be open and the user may then remove themicroplate. Method 1200 then ends.

In this way, a controlled dose of germicidal UV may be delivered througha modular light engine including an array of light emitting diodesconfigured to direct germicidal UV to a microplate inside chamber of amicroplate irradiation system.

In one example, the modular light engine may deliver a dose ofgermicidal light comprising multiple wavelengths (two or more) forinactivation. The multiple wavelengths used for inactivation may beselected based on contaminants to be inactivated, for example UVC lightat 255 nm may target nucleic acids, UVC light at 275 nm may targetprotein stability by specifically targeting cysteine and aromatics, andUVA light at 365 nm may target lysine in proteins (e.g., enzymes such asRNase A). In some examples, certain contaminating microorganisms maydemonstrate recovery (reactivation) after UV light exposure over time.In order to prevent this reactivation and to ensure an efficient andcomplete inactivation, two or more wavelengths of germicidal UV may beused. Additionally, wavelengths of light outside of the UV range mayalso be emitted (along with UV light, at least in some examples). Forexample, infrared (IR) light (e.g., at 1640 nm) may result inmelting/disassociation of alpha helices while visible light (e.g., at405 nm) may target common biological pigments (e.g., produced bymicroorganisms and therefore targeting such microorganisms).

Referring now to FIG. 13, a second embodiment of a bio-inactivationdevice is shown. The bio-inactivation device 1300 may be configuredsimilarly to the bio-inactivation device of FIG. 1 but may be used as acompact hand-held illumination unit for surface disinfection without themicroplate system. Such a device may be used as a point-of-use devicefor disinfecting surfaces and materials in one example. In anotherexample, the bio-inactivation device of FIG. 13 may be incorporated in alarger light emission system for bio-inactivation applications (e.g.,disinfection, DNA amplification, and sequencing). The device may behoused in housing 1301 and may include a control system 1326 (e.g., aprocessor and memory storing instructions executable by the processor),a modular light engine 1302 with one or more light emitting devices(e.g., LEDs) 1306, one or more user interfaces such as user interface1330 (e.g., a mouse, keyboard, touch screen, display menu), and acommunication system 1328 operable to couple the controller to one ormore remote computing devices, for example.

The controller of the control system 1326 may be an electroniccontroller and may include a memory storing instructions executable tocarry out one or more of the methods described herein. The controllermay include one or more physical logic devices, such as one or moreprocessors, configured to execute instructions. Additionally oralternatively, the controller may include hardware or firmwareconfigured to carry out hardware or firmware instructions. The memorymay include removable and/or built-in devices, including optical memory,solid-state memory, and/or magnetic memory. The memory may include;volatile, nonvolatile, static, dynamic, read/write, read-only,random-access, sequential-access, location-addressable,file-addressable, and/or content-addressable devices. The memory andlogic device(s) may be integrated together into one or morehardware-logic components, such as field-programmable gate arrays(FPGAs). The control system 1326 may control the activation status(e.g., on or off) as well as the intensity of light emitted from eachlight emitter via coupling electronics 1322.

Bio-inactivation device 1300 may include a modular light engine 1302comprising an array of light emitters 1306, such as light emittingdiodes 1320 (LEDs), for example, or OLEDs, plasma discharge, or otherlight emitters. Each light emitter or group of emitters may be coupleddirectly or indirectly to power source 1324. Each light emitter or groupof emitters may be coupled (e.g., mounted or bonded) to a substrate1308. Substrate 1308 may further be coupled to a thermal device 1316,which may be an active thermal regulation system, such as a Peltierdevice, or may be a passive thermal regulation system, such as aheatsink. Modular light engine 1302 may be configured similarly tomodular light engine 12 and/or 220, and as such includes a plurality oflight emitting devices bonded to a substrate (e.g., comprising an arrayof LEDs), which is coupled to a heat sink with cooling fins. However,other configurations are possible.

A temperature sensor 1310 is shown coupled to substrate 1308 formeasuring a temperature of the substrate. In other examples, thetemperature sensor may be positioned to measure a temperature of one ofthe light sources directly and/or additional temperature sensors may bepresent. Output from temperature sensor 1310 may be used to controlthermal device 1316 and/or the intensity of the light emitter(s) 1306.For example, if the output from the temperature sensor indicates thatthe light emitters are greater than a threshold temperature, the thermaldevice (if an active thermal device) may be activated to cool the lightemitters and/or the intensity of light output by the light emitters maybe decreased, to avoid degradation to the light emitters and/or othercomponents. In yet other examples, temperature sensor 1310 may beomitted or coupled to a different component of the inactivation system.

The light emitted by one or more light emitters 1306 may travel along alight path to a treatment surface 1314. In some examples, the lighttraveling along the light path may pass through light transfer optics1312 that may function to filter, focus, redirect, or otherwisecondition the light to produce a desired illumination pattern, beforereaching the treatment surface. The light transfer optics 1312 mayinclude a bandpass filter, lenses (e.g., ball lens, collimating lens,Fresnel lens), collimators, light guides, and/or other optics.

As described earlier, the light produced by the light emitters 1306 maycomprise multiple wavelengths (two or more) being incident on thetreatment surface. The use of multiple wavelengths of light may extendthe number of possible inactivation targets. For example, 255 nm UVlight may target nucleic acids, 275 nm UV light may target proteinstability by specifically targeting cysteine and aromatics, 365 nm UVlight may target lysine in proteins (e.g., enzymes such as RNase A),while 405 nm visible light may target common biological pigments (e.g.,produced by some microorganisms). As will be explained in more detailbelow, the use of multiple wavelengths at one time may be synergistic,as the effect of the combined wavelengths is greater than the sum of theeffects of the individual wavelengths. As used herein, the term“multiple wavelengths of light” may refer to multiple peak or averagewavelengths of light. For example, a light emitter configured to outputlight at 275 nm may actually output light at 275 nm plus light in awavelength range around 275 nm, such as light from 270-280 nm. Likewise,a light emitter configured to output light at 365 nm may actually outputlight at 365 nm plus light in a wavelength range around 365 nm, such as360-370 nm. As such, when reference is made to multiple wavelengths oflight herein, the multiple wavelengths may be different peak or averagewavelengths output from different light emitters, for example.

In one example, a specified excitation wavelength of light (λ EX, 1332of single or multiple wavelengths) may be emitted from the modular lightengine of the bio-inactivation device that may be incident on treatmentsurface 1314. When illuminated with an excitation source of a specifiedwavelength 1332, organisms, proteins, and/or other contaminants presenton the surface may exhibit fluorescence. Fluorescence refers tomolecular absorption of light at a first wavelength and its nearlyinstantaneous re-emission at a second, longer wavelength. For example,light of the excitation wavelength emitted by light emitters 1306 may beabsorbed by organisms on the illuminated surface causing electrons inthe organism to move to a higher energy, excited state. As the electronsrecede back down to their lower energy, ground state, energy is releasedas a photon of light at the second, longer wavelength. The amount andthe wavelength of the fluorescence emitted by the organisms may dependupon the molecular makeup of the cell (e.g., type of microorganism) andthe wavelengths of light used for excitation/illumination. Upon exposureto germicidal wavelengths of UV, IR, and/or visible radiation, themolecular makeup of a cell including its nucleic acids are irreversiblyaltered causing the level of fluorescence to decrease. In one example,when the treatment surface and hence any contaminating microorganismsand/or molecules are exposed to multiple germicidal wavelengths ofradiation targeting different aspects of an organism (e.g., proteins,nucleic acids, etc.), the level of fluorescence may be seen to decrease.

Thus, in order to measure the fluorescence (e.g., intensity level andwavelength of the emitted light), one or more signal detectors may beincluded in the bio-inactivation device that may be configured to detectlight emission at the second, longer wavelength emission λ EM_(1334) asfluorescence. In one example, the fluorescence signal detector may be aphotodetector 1318 (e.g., semiconductor photodiode). Thebio-inactivation device may be coupled to photodetector 1318 which maybe housed within the bio-inactivation device and further becommunicatively coupled to the control system. During bio-inactivationdevice operation, photodetector 1318 may convert a detectedamount/intensity and wavelength of emitted light into electrical currentand transmit it to control system 1326 as a measure of fluorescence fromsurface 1314. In some examples, the incident and/or emitted light mayinclude a spectrum of wavelengths (two or more wavelengths) and it maybe desirable to detect a range of wavelengths. Additionally, thefluorescence signal detector may comprise a photodetector with anintegral filter to reduce unwanted spectral emission (e.g., noise fromwavelengths outside the desirable range of wavelengths being measured),and enhance the signal-to-noise ratio (SNR).

By measuring the change in fluorescence using the photodetector (beforeand after multi-wavelength light exposure), the bio-inactivation deviceof FIG. 13 may enable the user to determine the presence and amounts ofmicroorganisms on surface 1314. Fluorescence from the surface may bemeasured by the photodetector prior to germicidal light exposure, andfluorescence information may be transmitted to and stored in the memoryof the control system. The surface may then be exposed to germicidallight of a desired intensity, desired dose, and desired pattern ofirradiation for a specified duration of time. In one example,irradiation parameters may be selected by a user using a menu on adisplay screen coupled to the bio-inactivation device, e.g., userinterface 1330, or may be received via an external device, e.g.,external device 1328. Following light exposure, fluorescence from thetreated surface may be detected and measured by the photodetector andthe information relayed to the control system. The control system maythen compare the fluorescence measurements taken prior to and afterlight treatment of the treatment surface and assess if inactivation ofcontaminants on the surface was achieved.

In some examples, the output from the photodetector may be monitoredcontinuously (or nearly continuously) during the emission of thegermicidal light to track the inactivation of the microorganisms and/ormolecular contaminants on the treatment surface. The germicidal lightemission may be deactivated once the detected fluorescence meets athreshold condition for the surface contaminants (e.g., drops below aninactivation threshold).

While the multiple wavelengths of light output from the modular lightengine and fluorescence feedback control were described above withrespect to a hand-held device, other configurations are possible. Forexample, the bio-inactivation device in the form of a microplateirradiation system 10 including a modular light engine 12 of FIG. 1 maybe configured similarly to the modular light engine 1302 of thebio-inactivation device 1300 of FIG. 13, such that multiple wavelengthsof light may be output from modular light engine 12 to a microplate.Further, microplate irradiation system 10 may include a photodetector,similar to photodetector 1318, in order to measure fluorescence emittedby microorganisms on a surface of the microplate and adjust the lightintensity, duration of exposure, etc., based on the fluorescence asdescribed in more detail below. In alternative embodiments, thebio-inactivation device may be part of a lighting system of a tissueculture hood or other work space, a portable hollow unit, e.g., a boxfitted with UV-emitting LEDs, such that materials to be sterilized(e.g., remote controls, keys, cordless phones, cell phones, etc.) may bedropped inside the box for a defined period of time or other device.

FIG. 15 illustrates a method 1500 for determining bio-inactivation usingmulti-wavelength germicidal light based on fluorescence using abio-inactivation device, such as the bio-inactivation device of FIG. 13.In one example, method 1500 may be used to operate the bio-inactivationdevice of FIG. 1 including the microplate irradiation system.Instructions for carrying out method 1500 may be executed by acontroller, for example, the control system 1326 of FIG. 13 or thecontroller 14 of FIG. 1, based on instructions stored in the memory ofthe controller and in conjunction with signals received at thecontroller.

At 1502, method 1500 begins by illuminating a surface with an excitationlight source of a specified wavelength λ EX (single or multiplewavelengths of excitation). The surface being illuminated may be anysurface on which the bio-inactivation device may be positioned. Theexcitation source may comprise of one or more desired wavelengths ofillumination such that when emitted back from the surface at a higherwavelength, a level of fluorescence emanating from the surface to betreated may be obtained.

At 1504, method 1500 may measure an initial fluorescence level based onan amount and wavelength of light emitted from surface illuminated withexcitation source (λ EM) using a photodetector, e.g., photodetector 1318of FIG. 13. The initial fluorescence may be detected and measured by thephotodetector of the bio-inactivation device prior to treatment of thesurface with multi-wavelength germicidal radiation. The measured initialfluorescence information may be relayed and stored in the memory of thecontroller.

At 1506, method 1500 may receive irradiation cycle parameters. Theirradiation cycle parameters may include a desired intensity ofirradiation, a desired dose, a desired pattern of irradiation, and/or aduration of exposure for which the surface to be treated is to beexposed to germicidal irradiation. In one example, the irradiation cycleparameters may be received via user input, as indicated at 1508, forexample the user may select the irradiation parameters from a menu on adisplay screen. In another example, the irradiation cycle parameters maybe received through a port coupled to an external device, as indicatedat 1510. For example, the parameters may be pre-set by a user and may bestored on an external device e.g., a USB drive.

Additionally, as indicated at 1511, the method may include determiningconstituent wavelengths to be used for bio-inactivation based on targetsurface contaminants. The multiple constituent wavelengths selected maydepend upon the target(s) to be bio inactivated. The target(s) may be anenzyme in one example (e.g., RNase A) and the optimal wavelengthsselected for inactivation may be further based on the relativeabsorption characteristics of the target (e.g., RNase A enzyme) and itschemical bonds being targeted. Furthermore, microorganisms may differ intheir cellular makeup, for example some microorganisms may showsusceptibility to specific wavelengths of light on account of possessinga certain subset of compounds that may be targeted by the specificwavelength of light, while being resistant to others. For example, 255nm UV light may target nucleic acids, 275 nm UV light may target proteinstability via targeting cysteine and aromatics, 365 nm UV light maytarget lysine in proteins, while 405 nm visible light may target commonbiological pigments. In one example, a combination of UV light at 275 nmand 365 nm may be selected for inactivation while in another example acombination of UV at 255 nm and 275 nm may be chosen. The optimalconstituent wavelengths selected may therefore be based on priorknowledge of the type of organisms present on the contaminated surface,and therefore their cellular makeup for example. The target surfacecontaminants may be determined based on the received irradiation cycleparameters, e.g., a user may enter an input specifying the targetsurface contaminants.

Once the irradiation cycle parameters have been selected, method 1500may proceed to 1512 to operate the bio-inactivation system with desiredradiation intensity, radiation pattern, and/or exposure duration basedon irradiation cycle parameters set at 1506 above. The bio-inactivationdevice may irradiate a surface to be treated with germicidal light withthe selected irradiation cycle parameters described above. Operating thebio-inactivation system may include activating one or more lightemitters, such as the LEDs described above, at the specified intensity,pattern, duration, and wavelength(s). In examples where multiplewavelengths of light are to be emitted, a first subset of LEDs may becontrolled to output light of a first wavelength (e.g., 275 nm) while asecond subset of LEDs may be controlled to output light of a secondwavelength (e.g., 365 nm). To ensure even coverage of both wavelengthsof output light across the entire treatment surface, the differentsubsets of LEDs may be interweaved, for example the first subset of LEDsmay alternate rows with the second subset of LEDs, alternate columnswith the second subset of LEDs, etc.

Method 1500 may detect inactivation by measuring the fluorescence levelafter exposure, based on an amount and wavelength of emitted light fromsurface λ EM using a photodetector at 1514. As mentioned earlier withreference to FIG. 13, an amount and wavelength of emitted light(measured as fluorescence) may be based on the molecular makeup ofbiological organisms and other contaminants present on the surface. Uponexposure to light in the germicidal wavelength range, molecularcontaminants present on the surface or within cells of organisms presenton the surface may be irreversibly altered/disrupted, leading to loss ordecrease of fluorescence. Thus, the photodetector may measure a level offluorescence of the surface after treatment and fluorescence informationmay be relayed to the controller in real time, allowing a real timeestimation of the reduction in fluorescence indicative of inactivationof molecular contaminants or microorganisms.

At 1516, method 1500 may determine if the change in fluorescence level(initial vs. after exposure) is greater than a pre-determined threshold.The threshold at 1516 may be a non-zero threshold and may represent achange in fluorescence value above which inactivation of contaminantsand/or microorganisms may be successfully achieved. The change influorescence may be calculated by the controller. The controller maycalculate a difference (e.g., change) based on initial fluorescence(obtained at 1504 above and stored in the memory of the controller) andfluorescence after germicidal light exposure (from 1514).

If the change in fluorescence is not determined to be greater than thethreshold (e.g., NO at 1516), method 1500 may adjust thebio-inactivation system operating parameters (e.g., adjust radiationintensity, radiation pattern and/or exposure duration) at 1518. In oneexample, a change in fluorescence not greater than the threshold mayindicate that inactivation with the selected radiation parametersincluding the multiple wavelengths was unsuccessful (e.g.,microorganisms and/or other contaminants were not successfullyinactivated). In another example, a change in fluorescence below thethreshold may indicate partial inactivation with the selected radiationparameters (e.g., microorganisms and/or other contaminants were notcompletely inactivated or all microorganisms and contaminants were notinactivated). In one example, the bio-inactivation system operatingparameters of radiation intensity, pattern, and/or duration of exposuremay be adjusted (e.g., the duration may be increased in one example). Inanother example, alternative multiple wavelengths or additionalwavelengths targeting other aspects of the microorganisms may beselected and method 1500 be carried out again.

For example, a first fluorescence response may indicate inactivation ofone type of microorganism (e.g., bacteria) while a second fluorescenceresponse may indicate incomplete inactivation of a different type ofmicroorganism (e.g., virus). Accordingly, if the first fluorescenceresponse is observed (e.g., a first change in fluorescence level at agiven excitation wavelength), it may be determined that bacteria on thetreatment surface have been inactivated. If a second fluorescenceresponse is also observed (e.g., a second change in fluorescence at adifferent excitation wavelength), it may be determined that virus on theinactivation surface has been only partially inactivated. At that point,the system may change the wavelength(s) and/or intensity of light outputfor viral inactivation. Method 1500 may then loop back to 1512 tooperate the bio-inactivation system with the desired wavelengths,radiation intensity, radiation pattern, and/or exposure duration basedon irradiation cycle parameters. In still further examples, the systemmay automatically determine target contaminants present on the treatmentsurface based on the detected fluorescence prior to output of thegermicidal light. For the example, the system may determine that bothbacteria and virus are present on the surface based on the initialdetected fluorescence. The system may then automatically select one ormore wavelengths of germicidal light to be output and may adjust thewavelength(s) and/or intensity of the germicidal light during the courseof the sterilization process to target all the identified contaminantson the surface. In still further examples, a first contaminant may beinitially detected and once the first contaminant has been fullyinactivated, the remaining fluorescence signal may indicate the presenceof a second, different contaminant still on the treatment surface.

Thus, the bio-inactivation device described herein may improveinactivation of different kinds of microorganisms by changing parameterswithin an irradiation cycle or dynamically adapting the irradiationparameters. As an example, a target surface may have a relatively largenumber of bacteria and a relatively small number of virus. The surfaceitself may be mildly susceptible to damage when exposed to light in theUV-A range, so a purely UV-C inactivation recipe may be selected (e.g.,the surface may be comprised of a plastic paraffin film or othermaterial prone to degrading when exposed to light in the UV-A range).The fluorescence emitted by the large number of active bacteria mayobscure the presence of the virus, until most of the bacteria areinactivated by the UV-C irradiation cycle and the viral load is able tobe detected. At that point, the bio-inactivation device may prompt theuser to select a more effective virus-inactivating recipe, or thebio-inactivation device may be configured to automatically enable ashort, dual-wavelength UV-A/UV-C cycle that is more effective againstvirus. Accordingly, the treatment surface material composition may alsobe taken into account when selecting appropriate irradiation cycleparameters.

On the other hand, if the change in fluorescence is determined to begreater than the threshold (e.g., YES at 1516), method 1500 may move to1520 to display inactivation data to user. In one example, inactivationdata may include a level of inactivation as determined by the change influorescence (e.g., percentage change). Method 1500 may continue to 1522to deactivate the modular light engine of the bio-inactivation device,by deactivating the LEDs for example. Method 1500 then returns.

In this way, bio-inactivation of microorganisms based on real timefluorescence feedback using the bio-inactivation device may bedetermined and operating radiation parameters may be accordinglyadjusted to achieve complete and rapid inactivation.

Referring now to FIG. 14, a third embodiment of a bio-inactivationdevice is shown. The bio-inactivation device 1400 may be configuredsimilar to the bio-inactivation device of FIG. 1 but may be used as acompact hand-held illumination unit for surface disinfection without themicroplate system, similar to the embodiment shown in FIG. 13. Such adevice may be used at a point-of-use for disinfecting surfaces andmaterials and may be optionally incorporated in larger light emissionsystem for bio-inactivation applications. Device 1400 may be housed inhousing 1401 and may include a control system 1426 (e.g., including aprocessor and memory), a modular light engine 1402 with one or morelight emitting devices (e.g., LEDs) 1406, one or more user interfacessuch as user interface 1430 (e.g., a mouse, keyboard, touch screen,display menu), and a communication system 1428 operable to couple thecontroller to one or more remote computing devices, for example. Thecontroller of the control system 1426 may be an electronic controllerand may include a memory, storing instructions executable to carry outone or more of the methods described herein. The control system 1426 maycontrol the activation status (e.g., on or off) as well as the intensityof light emitted from each light emitter via coupling electronics 1422.

Bio-inactivation device 1400 may include a modular light engine 1402comprising an array of light emitters 1406 that may be light emittingdiodes 1420 (LEDs), for example. Each light emitter may be coupleddirectly or indirectly to power source 1424 and may be coupled to asubstrate 1408. Substrate 1408 may further be coupled to a thermaldevice 1416, which may be an active or a passive thermal regulationsystem. A temperature sensor 1410 is shown coupled to substrate 1408 formeasuring a temperature of the substrate. In other examples, thetemperature sensor may be positioned to measure a temperature of one ofthe light sources directly and/or additional temperature sensors may bepresent. Output from temperature sensor 1410 may be used to controlthermal device 1416 and/or the intensity of the light emitter(s) 1406.In yet other examples, temperature sensor 1410 may be omitted or coupledto a different component of the inactivation system.

The light emitted by one or more light emitters 1406 may travel along alight path to a treatment surface 1414. In some examples, the lighttraveling along the light path may pass through light transfer optics1412 including a filter, lenses and/or other optics that may function tofilter, focus, redirect, or otherwise condition the light to produce adesired illumination pattern. As described earlier, the light producedby the light emitters 1406 may comprise multiple wavelengths (two ormore) being incident on the treatment surface. The emission of multiplewavelengths of light may extend the number of possible inactivationtargets, for example 255 nm UV light may target nucleic acids, 275 nm UVlight may target protein stability by specifically targeting cysteineand aromatics, 365 nm UV light may target lysine in proteins while 405nm light may target common biological pigments (e.g., produced by somemicroorganisms). Further, the use of multiple light wavelengths at onetime may be synergistic, as the effect of the combined wavelengths isgreater than the sum of the effects of the individual wavelengths.

In one example, a specified wavelength of light λ₀ may be emitted fromthe modular light engine of the bio-inactivation device that may beincident (I₀) on treatment surface 1414. When illuminated with anexcitation source of a specified wavelength λ₀, living (e.g., intact)microorganisms present on the surface may reflect the light differentlywhen compared to a dead (e.g., inactive) microorganisms. Typically, deadmicroorganisms lose their cellular integrity (e.g., cell membrane, cellwall, or coat integrity may be disrupted), allowing their contents tospread out on the surface, and may therefore reflect light differentlythan their living counterparts. This spreading of cellular contents maybe detectable as a change in optical properties (e.g., a decrease or anincrease in reflectance) of the surface. In one example, when thesurface contaminants are exposed to multiple germicidal wavelengths ofradiation targeting different aspects of an organism (e.g., proteins,nucleic acids, etc.), the reflectance may be seen to change.

In one example, the bio-inactivation device of FIG. 14 may use thiscriterion to assess a level of inactivation for a given surface. Forexample, a surface may be illuminated with light of a specificwavelength and an initial reflectance I based on an amount of reflectedlight may be measured. Germicidal irradiation parameters may then beselected. In one example, the germicidal irradiation parameters may beselected based on the amount of reflected light, e.g., the reflectedlight may indicate relative levels and/or types of contaminants on thetreatment surface. The surface may then be exposed to multiplewavelengths of germicidal light of a desired intensity, desired dose,and desired pattern of irradiation for a specified time duration. In oneexample, irradiation parameters including a combination of wavelengthsto be used for bio-inactivation may be selected using a menu on adisplay screen coupled to the bio-inactivation device, e.g., userinterface 1430, or may be selected via an external device, e.g.,external device 1428. The bio-inactivation device may be operated withthe selected parameters to inactivate microorganisms on the givensurface by UV and/or other light exposure. After exposure, the surfacemay once again be illuminated with light (e.g., of a specificwavelength, used before to measure initial reflectance) and areflectance after exposure to germicidal light may be measured. Based ona change in reflectance from before multi-wavelength light exposure toafter bio-inactivation with multi-wavelength light, a level ofinactivation of microorganisms on the surface may be evaluated.

In order to measure an amount of reflected light, one or more signaldetectors with an optional filter may be included in thebio-inactivation device 1400 configured to detect reflectance before andafter germicidal light exposure from surface 1414. In one example, thereflectance detector may be a photodetector (e.g., semiconductorphotodiode with an optional integrated filter), such as photodetector1418. In one example, an additional photodetector configured to measurefluorescence may also be present. The photodetector may be housed withinthe bio-inactivation device and be communicatively coupled to thecontrol system. During bio-inactivation device operation, photodetector1418 may convert a detected amount of reflected light into electricalcurrent and transmit it to control system 13426 as a measure ofreflectance from surface 1414.

In this way, following light exposure, reflectance from the treatedsurface may be detected and measured by the photodetector and theinformation relayed to the control system. The control system may thencompare the reflectance measurements taken prior to and after lighttreatment of the treatment surface and assess if inactivation ofcontaminants on the surface was achieved.

In some examples, the output from the photodetector may be monitoredcontinuously (or nearly continuously) during the emission of thegermicidal light to track the inactivation of the microorganisms and/ormolecular contaminants on the treatment surface. Once the detectedreflectance meets a condition relative to a threshold (e.g., changesbeyond a threshold indicating inactivation of the contaminants on thetreatment surface), the germicidal light emission may be deactivated.

While the multiple wavelengths of light output from the modular lightengine and reflectance feedback are described above with respect to ahand-held device, other configurations are possible. For example, thebio-inactivation device in the form of a microplate irradiation system10 including a modular light engine 12 of FIG. 1 may be configuredsimilarly to the modular light engine 1402 of the bio-inactivationdevice 1400 of FIG. 14, such that multiple wavelengths of light may beoutput from modular light engine 12 to a microplate. Further, microplateirradiation system 10 may include a photodetector, similar tophotodetector 1418, in order to measure fluorescence emitted bymicroorganisms or other molecular contaminants on a surface of themicroplate and adjust the light intensity, duration of exposure, etc.,based on the reflectance as described in more detail below.

FIG. 16 illustrates a method 1600 for determining bio-inactivation basedon reflectance in accordance with the bio-inactivation device of FIG.14. In one example, method 1600 may be used to operate thebio-inactivation device of FIG. 1 including the microplate irradiationsystem. In another example, method 1600 may also operate thebio-inactivation device of FIG. 13. Instructions for carrying out method1600 may be executed by a controller, for example, the control system1426 of FIG. 14, control system 1326 of FIG. 13 or the controller 14 ofFIG. 1, based on instructions stored in the memory of the controller andin conjunction with signals received at the controller.

At 1602, method 1600 begins by illuminating a surface with an excitationlight source of a specified wavelength. The specified wavelength may beone that is selected based on a robust signal to noise ratio. Uponillumination of the surface (e.g., surface contaminated withmicroorganisms and/or other contaminants), light striking the surfacemay be reflected back to the bio-inactivation device in anamount/direction based on the presence, amount, and cellular integrityof the constituents of the surface. The light that is emitted for thereflectance measurement may be the same wavelength or a differentwavelength than the light emitted for sterilizing the treatment surface,as explained below.

At 1604, method 1600 may measure an initial reflectance Io based on anamount of reflected light from the surface using a photodetector. In oneexample, the amount of reflected light may be proportional to an amountof intact microorganisms present on the surface. The reflected light(e.g., reflectance) measured may include a constant reflectance from thesurface. The initial reflectance may be measured by the photodetector ofthe bio-inactivation device prior to the surface being exposed togermicidal radiation. This could be considered as background light,which may then be stored in controller memory.

At 1606, method 1600 may select irradiation cycle parameters. Theirradiation cycle parameters may include a desired intensity ofirradiation, a desired dose, a desired pattern of irradiation, and/or aduration of exposure for which the surface to be treated is to beexposed to germicidal irradiation. In one example, the irradiation cycleparameters may be received via user input, as indicated at 1608, forexample the user may select the irradiation parameters from a menu on adisplay screen. In another example, the irradiation cycle parameters maybe received through a port coupled to an external device, as indicatedat 1610. For example, the parameters may be pre-set by a user and may bestored on an external device, e.g., a USB drive.

Receiving the irradiation cycle parameters may also include determiningconstituent wavelengths (multiple) to be used for bio-inactivation basedon target surface contaminants, as indicated at 1611. The multipleconstituent wavelengths selected may depend upon the target to be bioinactivated, e.g., an enzyme such as RNase A. The optimal wavelengthsselected for inactivation may be further based on the relativeabsorption characteristics of the target (e.g., enzyme) and the chemicalbonds being targeted. Furthermore, microorganisms may vary in theircellular makeup. Some microorganisms may show susceptibility to specificwavelengths as a consequence of possessing a certain subset of compoundsthat may be targeted by the specific wavelength of light, while beingresistant to others. For example, 255 nm UVC light may target nucleicacids, 275 nm UVC light may target protein stability via targetingcysteine and aromatics, 365 nm UVA light may target lysine in proteins,while 405 nm visible light may target common biological pigments.

Once the irradiation cycle parameters have been selected, method 1600may proceed to 1612 to operate the bio-inactivation system with desiredradiation intensity, radiation pattern, and/or exposure duration basedon irradiation cycle parameters set at 1606 above. The bio-inactivationdevice may irradiate a surface to be treated with multiple wavelengthsof germicidal light with the selected irradiation cycle parameters.Operating the bio-inactivation system may include activating one or morelight emitters, such as the LEDs described above, at the specified LEDintensity, spatial pattern, time duration, and wavelength(s). Inexamples where multiple wavelengths of light are to be emitted, a firstsubset of LEDs may be controlled to output light of a first wavelength(e.g., 275 nm) while a second subset of LEDs may be controlled to outputlight of a second wavelength (e.g., 365 nm). Uniform spatial coverage ofall wavelengths may be ensured by alternating respective LEDs or groupsof LEDs in their positional layout (e.g., 275 nm column/row followed by365 nm column/row, etc.), comprising the overall group (i.e. array).

At 1614, method 1600 may illuminate the surface with the excitationlight source of a specified wavelength. As mentioned earlier in FIG. 14,an amount of reflected light (measured as reflectance) may be based onan amount of and the cellular integrity of microorganisms present on thesurface. Upon exposure to multiple wavelengths of light in thegermicidal range, microorganisms present on the surface may beinactivated, leading to loss of cellular integrity and therefore loss ofreflectance (e.g., decrease in reflectance). The subsequent variance inlight reflected from the cellular surface to the photodetector, andresulting change in photodetector signal (voltage) may be relayed to thecontroller, allowing a real time measure of the relative change inreflectance.

At 1616, method 1600 may measure reflectance after bio-inactivationbased on an amount of reflected light from the surface using aphotodetector. The reflectance may be proportional to an amount ofmicroorganisms present on the surface, which after bio-inactivation(e.g., due to UV exposure), may be expected to decrease. The measuredreflectance may also include a contribution from the surface, which maybe a constant value. The measured reflectance data afterbio-inactivation may be relayed to and stored in the memory of thecontroller. Method 1600 may calculate a change in reflectance (I₀−I) I)at 1618. The controller of the bio-inactivation device may perform thiscalculation based on the initial reflectance I₀ obtained at 1604 andreflectance I obtained at 1616 after exposing the surface withgermicidal light.

At 1620, method 1600 may determine if the change in reflectance (I₀−I)is greater than a threshold. The threshold at 1620 may be apre-determined non-zero threshold and may represent a change inreflectance above which inactivation of microorganisms on the surfacemay be successfully achieved. If the change in reflectance is notdetermined to be greater than the threshold (e.g., NO at 1620), method1600 may adjust the bio-inactivation system operating parameters (e.g.,adjust radiation intensity, radiation pattern and/or exposure duration)at 1622. In one example, a change in reflectance not greater than thethreshold may indicate that inactivation with the selected radiationparameters was unsuccessful (e.g., microorganisms were not successfullyinactivated). In another example, a change in reflectance not greaterthan the threshold may indicate partial inactivation with the selectedradiation parameters (e.g., microorganisms were not completelyinactivated or all microorganisms were not inactivated). In one example,the bio-inactivation system operating parameters of radiation intensity,spatial pattern, and/or duration of exposure may be adjusted (e.g.,increased in one example). In another example, alternative multiplewavelengths or additional wavelengths targeting other aspects of themicroorganisms may be selected and method 1600 be carried out again.

For example, as explained above with respect to FIG. 15, a givenreflectance (e.g., a given change in reflectance at a given wavelengthor intensity of light) may indicate full inactivation of onemicroorganism but not another (e.g., full inactivation of bacteria butpartial inactivation of virus). In such examples, the wavelength(s)and/or intensity of germicidal light may be adjusted. In anotherexample, the user may specify a low-intensity longer cycle for a more“gentle” treatment of sensitive materials. For such a cycle, a longduration of low-intensity blue light (405 nm) may be effective againstbacteria. However, if the sensors detect the presence of active virus(e.g., based on the reflectance signal), the user may elect to include ashort duration of high-intensity 275 nm light or 275 nm and 365 nm lightthat is more effective against virus. Method 1600 may then loop back to1612 to operate the bio-inactivation system with the desiredwavelengths, radiation intensity, spatial pattern, and/or exposureduration based on irradiation cycle parameters.

On the other hand, if the change in reflectance is determined to begreater than the threshold (e.g., YES at 1620), method 1600 may move to1624 to display inactivation data to user. In one example, inactivationdata may include a level of inactivation as determined by the change inreflectance (e.g., percentage change). Method 1600 may continue to 1626to deactivate the modular light engine of the bio-inactivation device,by deactivating the LEDs for example. Method 1600 then returns.

In this way, bio-inactivation of microorganisms based on real timereflectance feedback using the bio-inactivation device may be determinedand operating radiation parameters may be accordingly adjusted toachieve complete and rapid inactivation.

In one example, each light emitter of the bio-inactivation device ofFIG. 1, 13, or 14 may emit light of a single wavelength or variablewavelengths, as dictated by the controller. Most inactivation methodsand systems currently in use utilize a single UV wavelength to achieveinactivation of specific targets (e.g., inactivation of nucleic acidsmay be optimally performed at 255 nm, inactivation of enzymes may beperformed at 275 nm to target cysteine and aromatics, etc.). In somesituations, inactivated targets (e.g., enzymes, microorganisms),especially those inactivated by a single germicidal UV wavelength maydemonstrate recovery (reactivation) after UV exposure, over a period oftime.

An example of a ubiquitous molecular contaminant frequently found onsurfaces in the laboratory includes the ribonuclease enzyme proteinRNase A. RNase A is not only a very stable enzyme, highly resistant todenaturation (disruption of protein) but is also found as a frequentcontaminant during automated DNA sequencing and amplification. RNase A,even when found in trace amounts in reagents and buffers used forsequencing and amplification of DNA, may interfere with experimentationand compromise results; RNase A targets RNA so it also desirable toremove even trace amounts of RNase A when performing work with RNA.Diethylpyrocarbonate (DEPC) treatment is the most common method used toinactivate RNases in water and other reagents. However, DEPC treatmentis not only time consuming (e.g., may require sample with DEPC to sitovernight) but can also produce secondary chemicals not suitable forautomated DNA analysis. Thus, RNase A serves as a suitable target toascertain the validity of the bio-inactivation device using multiplewavelengths of germicidal light to achieve a complete and effectiveinactivation. In one example, a combination of UV light at 275 nm and365 nm wavelengths may be used to target RNase A. As previouslydescribed, 275 nm UVC light may target protein stability by specificallytargeting cysteine and aromatics while 365 nm UVA light may targetlysine in proteins. FIG. 17 shows RNase A enzyme activity measured asrelative fluorescence, when a surface contaminated with RNase A istreated with various intensities (irradiance) of UV light at 275 nmwavelength, for a specific time duration. The horizontal axis (x-axis)denotes time after exposure (to the UV light) and the vertical axis(y-axis) denotes RNase A enzyme activity as relative fluorescence. Line1702 shows a positive control, e.g., an intact and active RNase A, notsubjected to UV light exposure. Line 1714 shows a negative control,e.g., a sample containing no RNase A. Lines 1704, 1706, 1708, 1710, and1712 all show RNase A activity after a five minute exposure toincreasing intensities of UV irradiance as marked on the figure legend.

In order to test the ability of the bio-inactivation device topermanently inactivate RNase A, a contaminated surface with RNase A wasexposed to 275 nm UVC light emitted from the modular light engine of thebio-inactivation device. The duration of exposure was fixed at fiveminutes, with subsequent measurement of RNase A enzyme reactivationactivity (relative fluorescence), over a period of time as shown by thex axis in FIG. 17. Line 1704 shows significant RNase A activity (e.g.,the enzyme is functional with almost complete reactivation), after beingexposed for five minutes to 275 nm UVC light at 10% irradiance (63.5mW/cm²). When the irradiance of 275 nm UVC light was increased to 25%, adecrease in RNase A reactivation was observed (compared to 10%irradiance), as shown by line 1706. As the irradiance of 275 nm UVClight was increased to 50% and subsequently to 75%, RNase A reactivationsignificantly decreased indicating nearly complete enzyme inactivation(e.g., denaturation or chemical modification of the enzyme), with 50%irradiance (316.7 mW/cm²) successfully inhibiting enzyme activity, shownby lines 1708 and 1710, respectively. 275 nm UVC light at 100%irradiance (635.4 mW/cm²) was effective in achieving completeinactivation of RNase A, with no detectable enzyme reactivation whenassayed over a period of time as shown by line 1712, similar to thenegative control (line 1714).

As seen from line 1708, 50% irradiance of 275 nm UV light for a timeperiod of five minutes led to a significant decrease in RNase A enzymeactivity with nearly complete inactivation (e.g., denaturation of theenzyme). In one example, the dose of UV light may impact theinactivation at a given wavelength and irradiance, and may be controlledby either changing the irradiance (as seen in FIG. 17) or by changingduration of exposure (as shown in FIG. 18).

FIG. 18 shows RNase A enzyme activity when the surface with RNase A istreated over increasing durations of exposure with 275 nm UVC light at50% irradiance. The horizontal axis (x-axis) denotes exposure time (toUV light) and the vertical axis (y-axis) denotes RNase A enzyme activityin relative fluorescence units. Line 1802 shows a steady decrease inenzyme activity when a surface contaminated with RNase A is subjected toincreasing exposure durations (doses) of 275 nm UVC light at 50%irradiance. Changing (e.g., increasing) the exposure time to effectivelyprovide a single dose of UVC light at 275 nm at 50% irradiance may yieldthe same results as exposing the surface to UVC light at 275 nm with anirradiance level above 50% (e.g., 75%, 100%).

As previously described, some microorganisms may exhibit resistance to asingle (selected) wavelength of UV light. In some situations, certainmicroorganisms may even demonstrate recovery (reactivation) aftersufficiently long UV exposure over time, rendering the single UVwavelength exposure ineffective. Reactivation may depend upon the typeof microorganism and various environmental conditions such as light,temperature, etc. Such reactivation of microorganisms may also rely onenzymes and has been observed for various microorganisms such asbacteria, virus, several protein vectors, fungi, etc. Differentmicroorganisms comprised of different enzymes, different cell membranestructures, etc., may be susceptible to different wavelengths of light(e.g., a given microorganism may be susceptible at one wavelength oflight but resistant at another). Similarly, some macromolecules mayexhibit inactivation susceptibility at some wavelengths while showingresistance at others.

In order to ensure complete and efficient inactivation, two or morewavelengths of light targeting different structures or pathways withinorganisms may be utilized, leading to a synergistic effect as depictedby FIG. 19. Referring now to FIG. 19, a graph 1900 depicting thesynergistic effect of using multiple UV wavelengths on RNase A enzymeactivity is shown. The horizontal (X) axis denotes UV exposure time andthe vertical (Y) axis denotes relative RNase A enzyme activity based onfluorescence efficacy. Line 1908 shows a positive control (e.g., anintact and functional RNase A) unexposed to any UV light and thereforeshowing no affected enzyme activity. Line 1910 shows the relativefluorescence of RNase A after exposure to 365 nm UVA light (measuredirradiance level of 10.1 mW/cm²). Line 1902 shows the inactivation ofRNase A as a function of time when exposed to UVC light at 275 nm at˜50% intensity (a measured irradiance level of 316.7 mW/cm²). Line 1904shows the inactivation of RNase A as a function of time when exposed toUVC light at 275 nm at 100% intensity (measured irradiance level of635.4 mW/cm²). Line 1906 shows the inactivation of RNase A as a functionof time when exposed to a combination of wavelengths using 275 nm UVC at50% irradiance (316.7 mW/cm²) and 365 nm UVA (10.1 W/cm²).

As seen from graph 1900, RNase A activity gradually declined and theenzyme was completely inactivated after 15 minutes exposure with 275 nmUVC light at approximately a 50% irradiance level as seen from therelative fluorescence values depicted by line 1902. In contrast, RNase Awas completely inactivated after a one minute exposure with 275 nm UVClight at 100% irradiance. However, as shown and explained in FIG. 17,several microorganisms may demonstrate recovery (reactivation) over timefollowing UV exposure. In order to prevent such reactivation and toenable a more efficient and complete inactivation, two or morewavelengths of UV light may be utilized to target different aspects ofan organism or an enzyme (e.g., RNase A). As seen from line 1906 ofgraph 1900, concurrent exposure of RNase A with two UV wavelengths (i.e.275 nm and 365 nm) resulted in a more rapid inactivation. RNase A enzymewas completely inactivated after three minutes, as measured byfluorescence. The combination of wavelengths was successful in achievingcomplete inactivation at a lower irradiance (50%) of UVC light at 275nm. Furthermore, the combination of wavelengths yielded completeinactivation after three minutes, compared to 25 minutes when 275 nm UVClight at 50% irradiation was used (See FIG. 18, line 1802).

In this way, the use of two or more wavelengths of light together (e.g.,275 nm and 365 nm) allowed for a synergistic interaction, enablingfaster and complete inactivation. The effect of using combinedwavelengths concurrently was greater than the sum of the effects of eachwavelength sequentially as seen from FIGS. 18 and 19. The reducedintensities and exposure time enable an operational advantage byextending the lifetime of the bio-inactivation device.

The concurrent use of multiple UV and/or other wavelengths may enablemore efficient and complete inactivation of microorganisms and othercontaminants. Real-time reflectance and/or fluorescence feedback mayalso be used to affect adjustment of the operating parameters (e.g.,exposure time and/or intensity level), to achieve complete and rapidinactivation.

The technical effect of multi-wavelength light exposure by the variousdepicted embodiments of the bio-inactivation device includesirreversible inactivation of both molecular contaminants and biologicalorganisms. Operation of the bio-inactivating device may enablesterilization of reagents and disinfect laboratory surfaces, making themsuitable for reproducible and accurate high-throughput amplificationand/or sequencing methods. Additionally, it may further eliminate falsepositive results by increasing signal-to-noise ratio (SNR).

An example provides for a microplate irradiation system, including ahousing, a chamber within the housing and configured to house amicroplate; and a modular light engine positioned in the housing abovethe chamber, the modular light engine including one or more lightemitting devices configured to emit radiation directed to a top surfaceof the microplate when the microplate is positioned in the chamber. In afirst example, the chamber is formed in part by a drawer configured tomove in and out of the housing, the drawer comprising a stage to holdthe microplate. In a second example, which optionally includes the firstexample, each light emitting device comprises an array of light emittingdiodes coupled to a substrate. In a third example, which optionallyincludes one or more of each of the first and second examples, eacharray of light emitting diodes comprises a first set of light emittingdiodes evenly spaced in a first row and a second set of light emittingdiodes evenly spaced in a second row. In a fourth example, whichoptionally includes one or more of each of the first through thirdexamples, each array of light emitting diodes comprises exactly sixteenlight emitting diodes. In a fifth example, which optionally includes oneor more of each of the first through fourth examples, the modular lightengine comprises exactly seven light emitting devices. In a sixthexample, which optionally includes one or more of each of the firstthrough fifth examples, each substrate is coupled to one or more coolingfins. In a seventh example, which optionally includes one or more ofeach of the first through sixth examples, the housing includes a vent,and the system further includes a fan configured to circulate air fromthe vent and across the one or more cooling fins. In an eighth example,which optionally includes one or more of each of the first throughseventh examples, when the microplate is positioned in the chamber, eacharray of light emitting diodes is positioned intermediate the microplateand the respective one or more cooling fins. In a ninth example, whichoptionally includes one or more of each of the first through eighthexamples, each light emitting device comprises exactly three coolingfins. In a tenth example, which optionally includes one or more of eachof the first through ninth examples, the system further including acontroller storing non-transitory instructions executable to adjust oneor more of an intensity of light output by each light emitting diode anda duration of light output by each light emitting diode. In an eleventhexample, which optionally includes one or more of each of the firstthrough tenth examples, each light emitting diode is configured tooutput light of a single, common wavelength. In a twelfth example, whichoptionally includes one or more of each of the first through eleventhexamples, a first subset of light emitting diodes is configured tooutput light of a first wavelength and a second subset of light emittingdiodes is configured to output light of a second, different wavelength.

Another example provides a method of irradiating a treatment surfacewith a bio-inactivation device, including activating a first lightemitting device of the bio-inactivation device to emit germicidal lightof a first wavelength toward the treatment surface, and activating asecond light emitting device of the bio-inactivation device to emitgermicidal light of a second, different wavelength toward the treatmentsurface. In a first example, the first wavelength is 275 nm and thesecond wavelength is 365 nm. In a second example, which optionallyincludes the first example, the method further includes deactivating thefirst light emitting device and the second light emitting device basedon feedback from a photodetector of the bio-inactivation device.

An example provides a bio-inactivation device including a first lightemitting device configured to emit germicidal light of a firstwavelength; a second light emitting device configured to emit germicidallight of a second wavelength; a photodetector; and a controller storingnon-transitory instructions executable to: activate the first lightemitting device and the second light emitting device to emit light ofthe first wavelength and the second wavelength at a treatment surface;and deactivate the first light emitting device and second light emittingdevice based on output from the photodetector. In a first example, thephotodetector is configured to detect light reflected from the treatmentsurface, and wherein the instructions are executable to deactivate thefirst light emitting device and the second light emitting deviceresponsive to the output from the photodetector indicating that anamount of light reflected from the treatment surface has changed by athreshold amount. In a second example, which optionally includes thefirst example, the photodetector is configured to detect fluorescenceemitted from one or more contaminants on the treatment surface, and theinstructions are executable to deactivate the first light emittingdevice and the second light emitting device responsive to the outputfrom the photodetector indicating that an amount of fluorescence emittedfrom the one or more contaminants on the treatment surface has changedby a threshold amount. In a third example, which optionally includes oneor more or both of the first and second examples, the first lightemitting device comprises one or more light emitting diodes configuredto output light at 275 nm and the second light emitting device comprisesone or more light emitting diodes configured to output light at 365 nm.

As used herein, an element or step recited in the singular and proceededwith the word “a” or “an” should be understood as not excluding pluralof said elements or steps, unless such exclusion is explicitly stated.Furthermore, references to “one embodiment” of the present invention arenot intended to be interpreted as excluding the existence of additionalembodiments that also incorporate the recited features. Moreover, unlessexplicitly stated to the contrary, embodiments “comprising,”“including,” or “having” an element or a plurality of elements having aparticular property may include additional such elements not having thatproperty. The terms “including” and “in which” are used as theplain-language equivalents of the respective terms “comprising” and“wherein.” Moreover, the terms “first,” “second,” and “third,” etc. areused merely as labels, and are not intended to impose numericalrequirements or a particular positional order on their objects.

FIGS. 1-11, 13, and 14 show example configurations with relativepositioning of the various components. If shown directly contacting eachother, or directly coupled, then such elements may be referred to asdirectly contacting or directly coupled, respectively, at least in oneexample. Similarly, elements shown contiguous or adjacent to one anothermay be contiguous or adjacent to each other, respectively, at least inone example. As an example, components laying in face-sharing contactwith each other may be referred to as in face-sharing contact. Asanother example, elements positioned apart from each other with only aspace there-between and no other components may be referred to as such,in at least one example. As yet another example, elements shownabove/below one another, at opposite sides to one another, or to theleft/right of one another may be referred to as such, relative to oneanother. Further, as shown in the figures, a topmost element or point ofelement may be referred to as a “top” of the component and a bottommostelement or point of the element may be referred to as a “bottom” of thecomponent, in at least one example. As used herein, top/bottom,upper/lower, above/below, may be relative to a vertical axis of thefigures and used to describe positioning of elements of the figuresrelative to one another. As such, elements shown above other elementsare positioned vertically above the other elements, in one example. Asyet another example, shapes of the elements depicted within the figuresmay be referred to as having those shapes (e.g., such as being circular,straight, planar, curved, rounded, chamfered, angled, or the like).Further, elements shown intersecting one another may be referred to asintersecting elements or intersecting one another, in at least oneexample. Further still, an element shown within another element or shownoutside of another element may be referred as such, in one example.

Spatially relative terms, such as “inner,” “outer,” “beneath,” “below,”“lower,” “above,” “upper,” and the like, may be used herein for ease ofdescription to describe one element or feature's relationship to anotherelement(s) or feature(s) as illustrated in the figures. Spatiallyrelative terms may be intended to encompass different orientations ofthe device in use or operation in addition to the orientation depictedin the figures. For example, if the device in the figures is turnedover, elements described as “below” or “beneath” other elements orfeatures would then be oriented “above” the other elements or features.Thus, the example term “below” can encompass both an orientation ofabove and below. The device may be otherwise oriented (rotated 90degrees or at other orientations) and the spatially relative descriptorsused herein interpreted accordingly.

This written description uses examples to disclose the invention,including the best mode, and also to enable a person of ordinary skillin the relevant art to practice the invention, including making andusing any devices or systems and performing any incorporated methods.The patentable scope of the invention is defined by the claims, and mayinclude other examples that occur to those of ordinary skill in the art.Such other examples are intended to be within the scope of the claims ifthey have structural elements that do not differ from the literallanguage of the claims, or if they include equivalent structuralelements with insubstantial differences from the literal languages ofthe claims.

1. A microplate irradiation system, including: a housing; a chamberwithin the housing and configured to house a microplate; and a modularlight engine positioned in the housing above the chamber, the modularlight engine including one or more light emitting devices configured toemit radiation directed to a top surface of the microplate when themicroplate is positioned in the chamber.
 2. The system of claim 1,wherein the chamber is formed in part by a drawer configured to move inand out of the housing, the drawer comprising a stage to hold themicroplate.
 3. The system of claim 1, wherein each of the one or morelight emitting devices comprises an array of light emitting diodescoupled to a substrate.
 4. The system of claim 3, wherein each array oflight emitting diodes comprises a first set of light emitting diodesevenly spaced in a first row and a second set of light emitting diodesevenly spaced in a second row.
 5. The system of claim 3, wherein eacharray of light emitting diodes comprises exactly sixteen light emittingdiodes.
 6. The system of claim 3, wherein the modular light enginecomprises exactly seven light emitting devices.
 7. The system of claim3, wherein each substrate is coupled to one or more cooling fins.
 8. Thesystem of claim 7, wherein the housing includes a vent, and furthercomprising a fan configured to circulate air from the vent and acrossthe one or more cooling fins.
 9. The system of claim 7, wherein when themicroplate is positioned in the chamber, each array of light emittingdiodes is positioned intermediate the microplate and the respective oneor more cooling fins.
 10. The system of claim 7, wherein each of the oneor more light emitting devices comprises exactly three cooling fins. 11.The system of claim 3, further comprising a controller storingnon-transitory instructions executable to adjust one or more of anintensity of light output by each light emitting diode and a duration oflight output by each of the one or more light emitting diodes.
 12. Thesystem of claim 3, wherein each of the one or more light emitting diodesis configured to output light of a single, common wavelength.
 13. Thesystem of claim 3, wherein a first subset of light emitting diodes isconfigured to output light of a first wavelength and a second subset oflight emitting diodes is configured to output light of a second,different wavelength. 14-23. (canceled)
 24. A system, comprising: ahousing; a chamber within the housing and configured to house amicroplate; a modular light engine positioned in the housing above thechamber, the modular light engine including one or more light emittingdevices configured to emit radiation directed to a top surface of themicroplate when the microplate is positioned in the chamber; whereineach light emitting device of the one or more light emitting devicescomprises an array of light emitting diodes coupled to a substrate, eachof the array of light emitting diodes comprises a first set of lightemitting diodes evenly spaced in a first row and a second set of lightemitting diodes evenly spaced in a second row; and a controller storingnon-transitory instructions executable to adjust one or more of anintensity of light output by the first and second sets of light emittingdiodes and a duration of light output by the first and second sets oflight emitting diodes.
 25. The system of claim 24, wherein the first setof light emitting diodes is configured to output light of a firstwavelength and the second set of light emitting diodes is configured tooutput light of a second wavelength different than the first wavelength.26. The system of claim 24, wherein each substrate is coupled to one ormore cooling fins.
 27. The system of claim 26, wherein the housingincludes a vent, and further comprising a fan configured to circulateair from the vent and across the one or more cooling fins.
 28. Abio-inactivation device, comprising: a housing; a chamber within thehousing and configured to house a microplate; a modular light enginepositioned in the housing above the chamber, the modular light engineincluding a first set of light emitting diodes evenly spaced in a firstrow and a second set of light emitting diodes evenly spaced in a secondrow configured to emit radiation directed to a top surface of themicroplate when the microplate is positioned in the chamber; and acontroller comprising computer-readable instructions stored onnon-transitory memory thereof that when executed enable the controllerto: activate the first set of light emitting diodes of thebio-inactivation device to emit a germicidal light of a first wavelengthtoward a treatment surface; activate the second set of light emittingdiodes of the bio-inactivation device to emit a germicidal light of asecond wavelength different than the first wavelength toward thetreatment surface; and adjusting one or more of the first set of lightemitting diodes and the second set of light emitting diodes based onfeedback from a photodetector of the bio-inactivation device, includingadjusting one or more of the first wavelength, the second wavelength,and a radiation pattern of the germicidal light based on feedback fromthe photodetector.
 29. The bio-inactivation device of claim 28, whereinthe photodetector is configured to detect light reflected from thetreatment surface, and wherein the instructions further enable thecontroller to deactivate the first set of light emitting diodes and thesecond set of light emitting diodes in response to feedback from thephotodetector indicating that an amount of light reflected from thetreatment surface has changed by a threshold amount.
 30. Thebio-inactivation device of claim 28, wherein the photodetector isconfigured to detect fluorescence emitted from one or more contaminantson the treatment surface, and wherein the instructions further enablethe controller to deactivate the first set of light emitting diodes andthe second set of light emitting diodes in response to feedback from thephotodetector indicating an amount of fluorescence emitted from the oneor more contaminants on the treatment surface has changed by a thresholdamount.